Association of the CYBA, PPARGC1A, PPARG3, and PPARD gene variants with coronary artery disease and metabolic risk factors of coronary atherosclerosis in a Russian population

Abnormalities in lipid metabolism and enhanced oxidative stress are considered as major risk factors for coronary atherosclerosis. Functional genetic variations in genes whose products are involved in lipid metabolism and antioxidant defense could therefore modulate risk of coronary artery disease (CAD). In this study, we evaluate whether the PPARGC1A Gly482Ser, PPARG3 (−681)C/G, PPARD +294T/C, and CYBA +242C/T gene variants confer the risk of CAD in a Russian population. A total of 313 CAD patients and 132 controls with no clinical sign of CAD were studied. The polymorphic markers were tested using a TaqMan assay. Allele and genotype frequencies in CAD patients and controls were compared using the Yates χ² test. Association of the genetic markers with metabolic risk factors of arterial atherosclerosis was studied using the analysis of variance test and then adjusted for conventional risk factors in the multiple regression analysis. For CYBA +242C/T, both the allele T and genotype T/T showed significant association with higher risk of CAD (odds ratio =1.49 and 3.89, respectively). The allele C and genotype C/C of the +294T/C marker of PPARD were associated with increased risk of CAD providing an odds ratio of 2.12 and 2.78, respectively. The risk variants of CYBA +242C/T and PPARD +294T/C markers were associated with higher low-density lipoprotein cholesterol and increased total serum cholesterol, respectively. In conclusion, the CYBA +242C/T and PPARD +294T/C variants modulate risk of CAD through their associations with atherogenic serum lipid profiles.     

Detecting protein-protein interactions in vesicular stomatitis virus using a cytoplasmic yeast two hybrid system

Protein-protein interactions play an important role in many virus-encoded functions and in virus-host interactions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques to detect such interactions, it has a number of limitations, including a requirement for the proteins of interest to be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detect interactions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicating in the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactions involving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA (NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened against each other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein, nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties were encountered using the membrane associated matrix and glycoproteins. A human cDNA library was also screened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with N in this context. The system presented can be redesigned easily for studies in other less tractable NNS viruses.     

CD4(+)CD25(high)CD127(low) regulatory T cells in patients with stable angina and their dynamics after intracoronary sirolimus-eluting stent implantation

Rapamycin contributes to the expansion of regulatory T cells (Tregs) in vitro. We investigated CD4(+)CD25(high)CD127(low) Treg level dynamics as well as the major parameters of cell immunity and sCD25 and highly sensitive C-reactive protein (hsCRP) concentrations in the blood of patients after coronary stenting (CS) with sirolimus (rapamycin)-eluting stents (SES; n = 43). The relation between initial Treg values and the severity of coronary atherosclerosis was observed. Treg and sCD25 levels were increased 1 month after CS versus baseline values and versus data in the control group (coronary angiography [CA], n = 20). A positive correlation between Treg and sCD25 levels was reported, whereas no relation was observed with the length of SES implanted. HsCRP level was increased during the first 7 days and returned to baseline values 1 month after CS/CA. Treg content is lower in patients with multivessel CAD. Elevated levels of Tregs and sCD25 after SES implantation might occur because of the immunomodulating effect of rapamycin.     

Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2 ^R168X mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients.     

Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54

Certain aminoglycosides are capable of inducing "translational readthrough" of premature termination codons (PTCs). However, toxicity and relative lack of efficacy deter treatment with clinically available aminoglycosides for genetic diseases caused by PTCs, including cystic fibrosis (CF). Using a structure-based approach, the novel aminoglycoside NB54 was developed that exhibits reduced toxicity and enhanced suppression of PTCs in cell-based reporter assays relative to gentamicin. We examined whether NB54 administration rescued CFTR protein and function in clinically relevant CF models. In a fluorescence-based halide efflux assay, NB54 partially restored halide efflux in a CF bronchial epithelial cell line (CFTR genotype W1282X/F508del), but not in a CF epithelial cell line lacking a PTC (F508del/F508del). In polarized airway epithelial cells expressing either a CFTR-W1282X or -G542X cDNA, treatment with NB54 increased stimulated short-circuit current (I (SC)) with greater efficiency than gentamicin. NB54 and gentamicin induced comparable increases in forskolin-stimulated I (SC) in primary airway epithelial cells derived from a G542X/F508del CF donor. Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents observed in wild-type (Cftr+/+) mice, comparable to gentamicin. NB54 exhibited reduced cellular toxicity in vitro and was tolerated at higher concentrations than gentamicin in vivo. These results provide evidence that synthetic aminoglycosides are capable of PTC suppression in relevant human CF cells and a CF animal model and support further development of these compounds as a treatment modality for genetic diseases caused by PTCs.     

Apoptosis and proliferation differences between CD34+ and CD34- leukemic subpopulations in childhood acute leukemia

In view of the clinical and biological significance of leukemic heterogeneity we studied the efficacy of spontaneous apoptosis and cell cycle distribution in CD34+ and CD34 - leukemic subpopulations. Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) leukemic samples with CD34 heterogeneous expression were separated into CD34+ and CD34 - fractions using fluorescence activated cell sorting. Cell cycle distribution, and apoptosis of the sorted subpopulations were estimated. CD34+ leukemic subpopulations had lower ability to apoptosis than that of CD34 - fractions in 6 out of 8 ALL samples and in 4 out of 5 AML samples. CD34+ fractions showed a higher percentage of proliferating cells compared to CD34 - cells in T-lineage ALL. These differences may lead to a more resistant phenotype of one of the subpopulations and reappearance this population in relapse.